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J. Am. Mosq. Control Assoc.
Vol. 1, No. 4
LABORATORY BIONOMICS OF TRIPTEROIDES ARANOIDES
TAKAO OKAZAWAS MASAHIRO HORIO^ MOTOYOSHl MOGP, ICHIRO MIYAGP
AND SUPAT SUCHARIT"
ABSTRACT. Tripteroides aranoides was colonized in the laboratory. Total duration of the immature stages
was ca. 3 weeks at 28°C, L:D = 15.5:8.5 with an ample food supply. Retardation of 4th instar development was
observed in larvae fed on insufficient food. Females were autogenous for the first clutch of eggs and required a
blood meal for maturation of the second clutch. Mating was initiated in flight and copulation occurred on the
cage wall. Gravid females hovered in small oblique loops above water in bamboo cups, whereupon a white egg
appeared at the abdominal tip, which was propelled by the swing of the abdomen towards water surface. The
females propelled eggs in the same manner into small apertures (11x4 mm) bored in bamboo.
Tripteroides is one of the largest genera of the
tribe Sabethini but the biology and behavior of
the species in this genus have scarcely been
studied. Miyagi (1973) observed the durations
of the developmental stages and ovipositional
behavior of Tp. bambusa (Yamada) in the labo-
ratory. Mori (1976) described autogeny in this
species. Beaver (1979) made observadons on
the biology of 3 Tripteroides species in pitcher
plants. For the rest of this genus, bionomic in-
formation is virtually confined to descriptions
of the breeding sites and observations on the
attraction to humans at the time of collection
In this study we established a laboratory col-
ony of Tp. aranoides (Theobald) and observed
the length of the developmental stages, blood-
feeding, fecundity and mating and oviposi-
MATERIALS AND METHODS
Colony maintenance. The laboratory colony
wras established with ca. 60 larvae collected from
bamboo stumps in San Pa-Tong, south of
Chiang Mai, Thailand. Gravid females were
provided with a bamboo cup (ca. 10 cm diam
and ca. 8 cm height) containing 200 ml tap
water for oviposition. Hatched larvae were
transferred to a white plastic tray (20 x 30 x 5
cm) containing 1,000 ml tap water, and pow-
dered mouse pellets plus dry yeast. Adults were
maintained in screened cages (20 X 20 X 30 cm)
and provided with cotton balls soaked with a 2%
' Division of Parasitology, Department of Mi-
crobiology, Saga Medical School. Nabeshima, Saga
^ Department of Medical Zoology, School of
Medicine, University of Occupational and Envi-
ronmental Health, Kitakyusha 807, Japan.
^ Laboratory of Medical Zoology, School of Health
Sciences, Faculty of Medicine, University of the
Ryukyus. Nishiharamachi, Okinawa 903-01, Japan,
* Dept. of Medical Entomology, Faculty of Tropi-
cal Medicine, Mahidol University, Bangkok I04()0,
sugar solution. The colony was maintained and
the following observations made in an insectary
at 28»C, 75-85% RH and L:D= 15.5:8.5, unless
Length of the developmental stages and
REPRODUCTIVE PHYSIOLOGY. Three hundred
larvae per tray were reared under 2 different
conditions: one group was given 0.02 gm/day/
tray of food throughout larval life, the other 0.2
gm before and 0.5 gm/day/tray after the ap-
pearance of 4th instar larvae. The number and
stage of larvae were recorded daily and pupae
were checked for adult emergence. For larvae,
the median time for development was deter-
mined graphically from the 50% molting time.
Cumulative percentages of stages from egg to
pupa were plotted on graph paper daily. Points
of each developmental stage were connected
and the point at which the line crossed the 50%
level indicated 50% molting time. One half of
the adults emerging from each tray were given
a 5% sugar solution and the other half, a 0.1%
sugar solution. Females were dissected and ex-
amined for mature eggs 6 days after
Premating period for females was deter-
mined from insemination rates. Ten to 20
newly-emerged, 24, 48 or 72 hr-old females
were kept in the 20 X 20 x 30 cm cages for 24
hr with the same number of mature males. All
females were dissected and their spermathecae
were examined for insemination. The insemi-
nation ability of males was determined from
insemination rates of mature females kept for
24 hr with newly-emerged, 24- or 48 hr-old
The prefeeding periods of two groups of fe-
males were determined by the incidence of en-
gorgement among females offered a hairless
mouse for 1 hour every day after emergence.
One group was provided with a bamboo
oviposition cup soon after emergence, white the
other 3 weeks afterwards.
To examine the possibility of maturing a sec-
ond egg clutch without a blood meal, females
fed on an ample food diet during larval life
were allowed to lay eggs of the first clutch in a
bamboo cup. All the eggs laid were removed
J. Am. Mosq. Control Assoc.
and counted daily. Five days after the last eggs
were laid, parous females were divided into two
groups. Females which engorged on a hairless
mouse were provided access to a 0.1% sugar
solution. The other, a blood-starved group, was
given a 5% sugar solution. Six days later, all
females were dissected to examine their ovaries.
Egg resistance to desiccation. About
2,000 eggs laid on the same day were collected
on a filter paper and divided into 10 batches of
152-292 eggs. One control batch was immedi-
ately transferred to tap water and kept at SS'C
and L;D= 15.5:8.5. Nine experimental batches
were taken out of water 3 days after oviposition
and held for storage at 20°C, 80% RH and
L:D=12:12 for 4-50 days. Each batch was
transferred to tap water at a scheduled time and
larval hatch was examined daily for 35 days.
Unhatched eggs were dissected to confirm the
embryonic death. The hatch rate of the ex-
perimental batches was corrected by that of the
Mating and ovipositional behavior. Mat-
ing and ovipositional behavior was observed in
a screened cage or a glass box (20 x 20 x 30
cm). Some observations were facilitated with
Length of the developomental stages. The
duration of each developmental stage under
two different food regimens is presented in
Table 1 . The mean duration time from oviposi-
tion to adult emergence under an ample food
supply was ca. 3 weeks. The duration time for
males was a littie shorter than that for females.
The total length of the larval stage under the
insufficient food regimen was greaUy increased,
due mainly to retarded growth at the 4th instar.
Females required 20 days more than males for
Reproductive physiology. Some males
transferred sperm less then 24 hr after
emergence (Fig. 1). Females were refractory to
insemination 24 hr after emergence. Some were
inseminated during 24-48 hr after emergence,
and most all by 72 hr. Inseminated females
appeared to be refractory to subsequent copu-
Females could produce first clutch eggs to
maturity without a blood meal (Table 2). The
autogeny rate was higher, and the mean
number of matured eggs greater for females
fed on ample food during both larval and adult
stages than for those given insufficient food as
larvae or adults. When underfed during both
larval and adult stages, most females did not
produce mature eggs.
Rates of autogeny and numbers of mature
eggs were related to wing length (Fig. 2). Fe-
males with wing lengths less than 2.1 mm never
produced mature eggs. All females with wings
more than 2.6 mm long had mature eggs. Be-
tween these extremes the rate of autogeny and
number of mature eggs was positively corre-
lated with wing length. The average fecundity
(K) is given by the following equations:
Y =RZ = 202.9 IX» - 1170.82X^
+ 2253. 33X - 1446.84
R = 1.69X - 3.43
Z = 119.92X2
-448.66X + 421.45
where R is rate of autogenous females, Z is
number of mature eggs for autogenous females
and X is wing length.
Oviposition of the autogenous first clutch
began 4 days after emergence and lasted for ca.
Table 1. Duration of developmental stages and mortality of Tripteroides aranoides under two nutritional
(Larva + Pupa)
* Larvae were fed on 0.2 g before and 0.5 g after appearance of 4th instar.
** Ranges in parentheses.
*** Obtained by graphic method.
J. Am. Mosq. Control Assoc.
Vol. I, No. 4
24 48 72 96
TIME AFTER EMERGENCE (hOUR)
Fig. 1. Time required for insemination of Trip-
teroides aranoides. Ten to 20 females were dissected at
12 days (Fig. 3); that of the anautogenous sec-
ond clutch began 4 days after engorgement.
Females differed in their egg laying patterns:
some laid the majority of the clutch in one day,
while others laid small egg batches on different
Blood feeding activity started after the be-
ginning of oviposition. Females provided with a
bamboo oviposition cup upon emergence took
the first blood meal 6 days after emergence or 2
days after the first oviposition. More than 95%
of them took a blood meal within 15 days (Fig.
4a). Females not provided with an oviposition
cup rarely consumed blood before oviposition
(Fig. 4b). Dissection of females soon after taking
a blood meal revealed many mature eggs, which
indicates that even partial oviposition can re-
lease the blood feeding activity.
A blood meal was required for the matura-
tion of the second clutch of eggs. All 25 females
which consumed blood had mature eggs. The
Z = 119.92 X - <UI8.66 X + 421. «5
r > 0.579
R . 1.69 X - 3.43
r^ - 0.904
(2.08 < X < 2.60)
1.8 2.0 2.2 2.4 2.6
WING LENGTH (MM)
Fig. 2. Autogenous fecundity of Tripteroides
aranoides in relation to wing length. Adults were fed
on 5% sugar solution, a. Number of mature eggs
(only females with mature eggs), b. Rate of females
with mature eggs.
mean number of mature eggs was greater for
the anautogenous clutch (108.1 eggs) than for
the preceding autogenous clutch (63.7 eggs).
Without a blood meal, follicular development
for the second clutch stopped at lib or earlier
stages of Macan's system (1950) in 14 of 16
blood-starved females. The remaining 2 had a
small number of mature eggs (1 and 17 eggs). It
was uncertain whether those were eggs of the
second autogenous clutch or remnants of the
Egg reslstance to desiccation. Soon after
being laid, eggs could not be stored at 20°C and
Table 2. Effects of larval and adult nutrition on the expression of autogeny in Tripteroides aranoides.
* Percentages and means followed by the different letters were significantly different by x'^-test (per-
centages) or f-test (means) (P<0.01).
** For autogenous females.
J. Am. Mosq. Control Assoc.
DAYS AFTER EMERGENCE
10 15 20
DAYS AFTER ENGORGEMENT
Fig. 3. Egg laying of Tripteroides aranoides females individually maintained. Arrows indicate blood-feeding.
80% RH as all the eggs caved-in within a day.
Three days after being laid, eggs retained the
original shape after drying, but the percentage
of flattened eggs increased with time. The
hatching rate was above 70% for the first 16
days of preservation, and decreased sharply
thereafter (Fig. 5). A few eggs hatched after 35
days of drying, but none hatched after 40 days.
Mating behavior. Mating was initiated while
both sexes were in flight. A male dashed to a
flying female and caught her legs. Then the
male slid under the body of the flying female
and faced her. The couple flew to the cage wall
or to the floor and rested. As soon as connect-
ing his terminalia to that of the female, the male
released his grasp and turned his body down;
their bodies were then arranged in a straight
DAYS AFTER EfCRGENCE
line, with heads in opposite directions (Fig. 6a).
Turning of the male was sometimes finished
before reaching the cage wall or floor. In such
cases, both sexes beat their wings, but the
couple flew in the direction of the female.
Copulation continued for an average of 134.3
seconds (range 106-165, n=15) after joining
terminalia. The male beat his wings throughout
the copulation. When the legs of a copulating
male touched the cage floor, wing beating
sometimes stopped. Males often hovered above
bamboo cups awaiting females coming for
Fig. 4. Cumulative percentage of Tripteroides
aranoides females taking a blood meal. a. Females
given an oviposition cup soon after emergence, b.
Females given a cup 3 weeks after emergence.
Number of females observed was 43 for a, and 31 for
b. Bars show number of eggs laid per female. Arrows
indicate when a bamboo oviposition cup was given.
10 20 30 40
DAYS OF PRESERVATION AFTER DRYING
Fig. 5. Relation of hatching rate to duration of dry
condition. Eggs were taken out of water 3 days after
oviposition. Number of eggs observed was 152-292
J. Am. Mosq. Control Assoc.
Vol. 1, No. 4
OviPosiTioNAL BEHAVIOR. Females oviposited
in flight. When a dosed bamboo internode (12
cm diam and 12 cm height) with a lateral aper-
ture (11x4 mm) was offered, gravid females
flew around the bamboo internode. A female
slowly approached the aperture with her pro-
boscis directed downwards and antennae
towards the aperture (Fig. 6b-ieft). In front of
the aperture the female hovered in a small ob-
lique loop several times, whereupon a single
white egg appeared at the tip of the abdomen.
Occasionally we observed females bearing 2 or
3 eggs. Hovering still for a moment at a distance
of 0.5— 1 cm from the aperture, the female
swung her abdomen towards the target (Fig.
6b-right). The egg was propelled 2.7-4.8 cm
horizontally into the aperture. Unless the fe-
male swung her abdomen, the egg remained
attached to the tip of the abdomen. Sometimes
females rested at the aperture with an egg on
the tip of the abdomen, but we have never ob-
served oviposition by females at rest on the
bamboo, nor did females enter into the bamboo
internode through the aperture.
When an open bamboo cup was provided,
gravid females hovered in oblique loops at a
distance of 2- 10 cm from the water surface,
whereupon an egg appeared. The female
propelled the egg while swinging her abdomen
towards the water surface. Some females faced
the inside wall of the bamboo cup at a distance
of 1-2 cm, and the projected eggs attached to
the moist bamboo wall.
In successive ovipositions, the female swung
her abdomen once every 10-30 seconds. Since
the number of the eggs laid in the bamboo cup
was the same as that of the swinging modon, it is
assumed that the female released an egg at each
time of swinging. When a bamboo internode
with an aperture was placed in the cage, the
female laid an average of 4.6 eggs (range 1-31,
nn = 98) in a single oviposition bout without
rest or without flying to any other part of the
cage from the oviposition site.
Reported lengths of immature stages in Trip-
teroides are 5-6 weeks for Tp. aranoides, Tp. bam-
husa and Tp. nepenthis (Edwards) collected from
Nepenthes in Malaysia (Beaver 1979), 4-6 weeks
for Tp. hisquamatus Lee (Assem 1959) and 20-29
days for Tp. hamhusa (Yamada) (Miyagi 1973).
These values were 1-2 weeks longer than the
present observations for Tp. aranoides supplied
ample food and reared at higher temperature.
The different food conditions and tempera-
tures may have produced different devel-
opmental times. Inadequate food prolonged
the larval stage in Tp. aranoides as in other mos-
quitospecies(Moeurandlstock 1980, Mori 1979,
Trpis 1979). Frank and Curtis (1977) found
that delay in development of Wyeomyia larvae
occurred principally in the 3rd instar under
subopdmal nutrition. In Tp. aranoides the de-
velopmental delay occurred mainly in the 4th
instar. Such instar specificity of developmental
delay has not been identified in the other Trip-
Some Tp. bambium females whose immature
stages were reared at 15°C and 10 h photophase
were autogenous, but at a higher temperature
and longer photophase, autogeny was inhibited
(Mori 1976). In contrast the rate of autogeny in
Fig. 6. Behavior of Tripleroides aranoides. a. Copulation. Upper mosquito is female and lower male. b. Oviposi-
tion into the aperture. Left female in approach and right in oviposition.
J. Am. Mosq. Control Assoc.
Tp. aranoides was very high, even under higher
temperature (28°C) and longer photophase
(15.5 h). This species may be autogenous for
the first egg clutch under natural conditions in
I stock et al. (1975) reported an effect of food
level on egg production in Wyeomyia smithii
Coquillett. The number of eggs produced is
greater for females fed on adequate food dur-
ing the larval stage than for those fed on a
limited quantity of food. Insufficient food at
the adult stage reduced the autogeny rate and
the egg number in Wyeomyia vanduzeei Dyar and
Knab (Nayar et al. 1979). Insufficient food
during the larval and/or adult stages affects the
autogeny rate and the number of eggs pro-
duced by Tp. aranoides. This means that the
carbohydrates which Tp. aranoides adults take
can be diverted to oogenesis, or that adults
consume nutrients stored during larval life.
In the Chiang Mai area the dry season con-
tinues for ca. 6 months (November-April),
during which bamboo stumps (the main
breeding site for Tp. aranoides) seem to dry up.
Tripteroides aranoides was found only in the rainy
season in Burma Qolly 1933) and Yunnan
(Chow 1949). Mattingly (1981) therefore in-
ferred that this species passes over the dry sea-
son in the adult or egg stage. Macdonald (1957)
reported the eggs of Tp. aranoides resistant to
desiccation for at least 10 days. In our study,
eggs could not withstand desiccation over 40
days under 80% RH, which is 20% higher than
the mean humidity in March and April at
Chiang Mai. We assume that Tp. aranoides con-
tinues to breed in concealed habitats such as
bored bamboos, in which sap accumulates even
in the dry season, rather than pass the dry sea-
son in the egg stage.
In the mating behavior of Tp. aranoides, two
successive steps were recognized; 1) contacting
terminalia face to face, and 2) copula with heads
in opposite directions. Mosquitoes with a short
duration of mating, e.g., Aedes aegypti (Linn.)
(Gwadz et al. 1971), usually complete insemina-
tion at the former step, the latter being absent.
Mosquitoes with a long duration, e.g., Aedes
iriomotensis Tanaka and Mizusawa (Miyagi and
Toma 1981), require the latter step for insemi-
The ovipositional behavior of Tp. aranoides is
similar to that of Sabethes chloropterus Humboldt
observed by Galindo (1958). Both species
oviposit in small vertical holes while flying.
Compared with the clutch size, the number of
eggs laid by Tp. aranoides females in a single
oviposition bout was fairly small. And females
often lay eggs of a clutch on different days. This
suggests that Tp. aranoides females deposit eggs
in small batches at several different breeding
places. Such low numbers of eggs in egg batches
may decrease competition among offspring
under limited food supply in small water col-
lections surfi as bamboo internodes.
We wish to express our gratitude to Prof. C.
Khamboonruang, Chiang Mai University, for
facilitating our research in Chiang Mai, Mr. T.
Deesin, Mahidol University, Mr. W. Choochote,
Chiang Mai University, Drs. M. Tsukamoto and
T. Toma, members of the overseas research
team, for their cooperation in collecting the
material, and to Prof. K. Tokaichi, Saga Medi-
cal School, for improving the English of die
manuscript. We also thank Ms. K. Noda, Saga
Medical School, for help in maintaining the col-
This study was supported by Grant-in-Aid
for Overseas Scientific Survey (No. 58041058),
the Ministry of Education, Sciences and Culture
of Japan (Leader, Prof. I. Miyagi).
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